Single Molecule Detection of Fluorescent Probes

这项研究将集中在开发一种新型的、超灵敏的荧光探针检测设备。将建立一个足够灵敏的检测系统，以观察单个荧光团发出的荧光。单分子计数或门控的新概念将被用来提供对罗利、拉曼和探测器背景的必要区分。为了开发这一检测系统，并展示对溶液中单个荧光分子的检测，我们将研究流动样品系统中荧光团的激光激发荧光。在该项目的第二部分，将利用该探测系统建立二维激光扫描仪系统，该系统将获得载玻片、凝胶和胶片等底物上单个荧光团的位置图像。多幅图像的自相关和互相关的概念将被用来提供对空间随机背景的进一步区分。对单个荧光团和薄膜中的荧光标记抗体或荧光珠的初步实验将用于演示成像技术。将进行可行性实验，探索这种新技术在凝胶或荧光抗体与细菌的结合上寻找标记蛋白质或DNA条带的敏感性。这一方法将提高分析灵敏度，这将特别有价值，因为它将提高免疫荧光分析和荧光检测DNA测序凝胶的速度和灵敏度。未来重要的应用可能包括单个细胞表面受体或标记的免疫荧光检测以及DNA探针在染色体上的定位。通过荧光标记检测单个细胞受体或标志物将使细胞和分子过程的研究人员能够更准确地了解这些过程。细胞的复杂分子组成导致很难从许多不同分子的背景中分离出特定的分子。检测的改进将使细胞生理学、神经生物学和生物物理细胞学等许多生物学领域受益。

This research will focus on the development of a novel and ultrasensitive apparatus for the detection of fluorescent probes. A detection system will be constructed that is sensitive enough to observe the fluorescence emitted by individual fluorophores. The new concept of single molecule counting or gating will be used to provide the necessary discrimination against Raleigh, Raman and detector background. To develop this detection system and to demonstrate the detection of individual fluorescent molecules in solution, the laser excited fluorescence from fluorophores in a flowing sample system will be studied. In the second part of this project, two-dimensional laser scanner systems will be constructed, utilizing this detection system, which will obtain images of the location of individual fluorophores on substrates such as slides, gels and films. The concepts of auto and cross correlation of multiple images will be used to provide further discrimination against spatially random background. Initial experiments on individual fluorophores and on fluorescent labeled antibodies or fluorescent beads in thin films will be used to demonstrate the imaging technique. Feasibility experiments exploring the sensitivity of this new technique for finding labeled protein or DNA bands on gels or fluorescent antibody conjugates with bacteria will be performed. The improved analytical sensitivity that will be provided by this methodology will be particularly valuable because it will increase the speed and sensitivity with which immunofluorescence assays and fluorescence-detected DNA sequencing gels can be performed. Significant future applications may include immunofluorescence detection of individual cell surface receptors or markers and the location of DNA probes on chromosomes. The detection of individual cell receptors or markers by fluorescent labeling will enable investigators of cellular and molecular processes to understand these processes more accurately. The complex molecular makeup of cells leads to difficulties identifying a specific molecule separately from a background of many different molecules. Detection improvement will benefit many areas of biology such as cell physiology, neurobiology, and biophysical cytology.